Assays and kits incorporating nucleic acid probes containing improved molecular switch

ABSTRACT

A probe for the detection of a nucleic acid target sequence containing a molecular switch comprising three essential elements: a probe sequence of 20-60 nucleotides surrounded by switch sequences of 10-40 nucleotides which are complementary to each other, wherein the state of the switch is useful for selectively generating a detectable signal if the probe is hybridized to a target; also, assays and kits utilizing such probes.

This is a divisional of copending U.S. application(s) Ser. No.07/251,696, filed Sep. 30, 1988, now U.S. Pat. No. 5,118,801.

This invention relates to the field of bioassays that involve nucleicacid hybridization probes. These bioassays are useful for the detectionof specific genes, gene segments or RNA molecules. The assays are usefulclinically, for, e.g., tissue, blood and urine samples, as well as infood technology, agriculture, and biological research.

BACKGROUND OF THE INVENTION

The use of nucleic acid hybridization probes for bioassays is wellknown. One of the early papers in the field directed to assays for DNAis Gillespie, D. and Spiegelman, S., A Quantitative Assay for DNA-RNAHybrids with DNA Immobilized on a Membrane, J. Mol. Biol. 12:829-842(1965). In general terms such an assay involves separating the nucleicacid polymer chains in a sample, as by melting, fixing the separated DNAstrands to a nitrocellulose membrane, and then introducing a probesequence which is complementary to a unique sequence of the materialbeing sought, the "target" material, and incubating to hybridize probesegments to complementary target segments, if targets are present.Non-hybridized probes are removed by known washing techniques, and thenthe amount of probe remaining is determined by one of a variety oftechniques outlined below which provides a measurement of the amount oftargets in the sample.

A more recently developed form of bioassay that uses nucleic acidhybridization probes involves a second probe, often called a "captureprobe." Ranki, M., Palva, A., Virtanen M., Laaksonen, M., and Soderlund,H., Sandwich Hybridization as a convenient Method for the Detection ofNucleic Acids in Crude Samples, Gene 21:77-85 (1983); Syvanen, A.-C.,Laaksonen, M., and Soderlund, H., Fast Quantification of Nucleic AcidHybrids by Affinity-based Hybrid Collection, Nucleic Acids Res.14:5037-5048 (1986). A capture probe contains a nucleic acid sequencewhich is complementary to the target, preferably in a region near thesequence to which the radioactively labeled probe is complementary. Thecapture probe is provided with a means to bind it to a solid surface.Thus, hybridization can be carried out in solution, where it occursrapidly, and the hybrids can then be bound to a solid surface. Oneexample of such a means is biotin. Langer, P. R., Waldrop, A. A. andWard, D. C., Enzymatic Synthesis of Biotin-Labeled Polynucleotides:Novel Nucleic Acid Affinity Probes, Proc. Natl. Acad. Sci. USA78:6633-6637 (1981). Through biotin the capture probe can be bound tostreptavidin covalently linked to solid beads.

The present invention is directed to the methods and means, includingassays and pharmaceutical kits containing requisite reagents and means,for detecting in an in vitro or ex vitro setting the presence of nucleicacid species.

It is a goal in this art to detect various nucleic acid sequences in abiological sample, in which the said sequences, as so-called targetsequences, are present in small amounts relative to its existenceamongst a wide variety of other nucleic acid species including RNA, DNAor both. Thus, it is desirable to detect the nucleic acid encodingpolypeptides that may be associated with pathological diseases orconditions, such as, for example, RNA of the human immunodeficiencyvirus. In addition to the detection of nucleic acids encoding theproteins of such viral particles, it is desirable to detect othernucleic acids characteristic of a pathological disease or condition suchas a defective gene, as in the case of hemophilia. It is also desirableto detect other nucleic acids whose presence in the sample indicatesthat the organism is able to resist the action of a drug, such as anantibiotic.

Several approaches have been used for detecting the probe. One is tolink a readily detectable reporter group to the probe. Examples of suchreporter groups are fluorescent organic molecules and ³² P-labeledphosphate groups. These detection techniques have a practical limit ofsensitivity of about a million targets per sample.

A second approach is to link a signal generating system to the probe.Examples are enzymes such as peroxidase. Probes are then incubated witha color-forming substrate. Leary, J. J., Brigati, D. J. and Ward, D. C.,Rapid and Sensitive Colorimetric Method for Visualizing Biotin-LabeledDNA Probes Hybridized to DNA or RNA Immobilized on Nitrocellulose:Bio-Blots, Proc. Natl. Acad. Sci. USA 80:4045-4049 (1983). Suchamplification reduces the minimum number of target molecules which canbe detected. As a practical matter, however, nonspecific binding ofprobes has limited the improvement in sensitivity as compared toradioactive tagging to roughly an order of magnitude, i.e., to a minimumof roughly 100,000 target molecules.

Yet another approach is to make many copies of the target itself by invivo methods. Hartley, J. L., Berninger, M., Jessee, J. A., Bloom, F. R.and Temple, G. S., Bioassay for Specific DNA Sequences Using aNon-Radioactive Probe, Gene 49:295-302 (1986). This can also be done invitro using a technique called "polymerase chain reaction" (PCR). Thistechnique was reported in Saiki, R. K., Scharf, S., Faloona, F., Mullis,K. B., Horn, G. T., Erlich, H. A., and Arnheim, N., EnzymaticAmplification of Beta-globin Genomic Sequences and Restriction SiteAnalysis for Diagnosis of Sickle Cell Anemia, Science 230:1350-1354(1985); Saiki, R. K., Gelfand, D. H. Stoffel, S., Scharf, S. J.,Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A.,Primer-directed Enzymatic Amplification of DNA With a Thermostable DNAPolymerase, Science 239:487-491 (1988); Erlich, H. A., Gelfand, D. H.,and Saiki, R. K., Specific DNA Amplification, Nature 331:461-462 (1988),and Mullis et al., European Patent Application Publication Nos. 200362and 201184 (see also U.S. Pat. Nos. 4,683,195 and 4,683,202). In PCR,the probe is complementary only to the beginning of a target sequencebut, through an enzymatic process, serves as a primer for replication ofan entire target. Each repetition of the process results in anotherdoubling of the number of target sequences until a large number, say, amillion copies, of the target are generated. At that point detectableprobes, e.g., radioactively labeled probes, can be used to detect theamplified number of targets. The sensitivity of this method of targetamplification is generally limited by the number of "false positivesignals" generated, that is, generated segments that are not true copiesof the target. Nonetheless, this method is quite sensitive. Theprocedure requires at least two nucleic acid probes and has three stepsfor a single cycle. This procedure is cumbersome and not alwaysreliable.

Yet another method for amplification is to link to the probe an RNA thatis known to be copied in an exponential fashion by an RNA-directed RNApolymerase. An example of such a polymerase is bacteriophage Q-betareplicase. Haruna, I., and Spiegelman, S., Autocatalytic Synthesis of aViral RNA In Vitro, Science 150:884-886 (1965). Another example is bromemosaic virus replicase. March et al., POSITIVE STRAND RNA VIRUSES AlanR. Liss, New York (1987). In this technique, the RNA serves as atemplate for the exponential synthesis of RNA copies by a homologousRNA-directed RNA polymerase. The amount of RNA synthesized is muchgreater than the amount present initially. This amplification techniqueis disclosed in Chu, B. C. F., Kramer, F. R., and Orgel, L. E.,Synthesis of an Amplifiable Reporter RNA for Bioassays, Nucleic AcidsRes. 14:5591-5603 (1986); Lizardi, P. M., Guerra, C. E., Lomeli, H.,Tussie-Luna, I. and Kramer, F. R., Exponential Amplification ofRecombinant-RNA Hybridization Probes, Bio/Technology 6:1197-1203(October, 1988), which is incorporated herein by reference and isattached hereto in manuscript form [hereinafter referred to as "Lizardiet al."]; published European Patent Application 266,399 (EP ApplicationNo. 87903131.8). After non-hybridized probes are removed by washing, theRNA polymerase is used to make copies of the replicatable RNA. Accordingto the disclosure of published European Patent Application No. 266,399,replication of the RNA may take place while the RNA is linked to theprobe. Alternatively, the replicatable RNA may be separated from theremainder of the probe prior to replication. That application alsodiscloses a variety of chemical links by which a probe sequence can bejoined to a replicatable RNA. In addition, it discloses that the probesequence may be part of a replicatable RNA, as described in Miele, E.A., Mills, D. R., and Kramer, F. R., Autocatalytic Replication of aRecombinant RNA, J. Mol. Biol. 171:281-295 (1983). That Europeanapplication also discloses that such recombinant RNAs must be able tohybridize specifically with the target sequence as well as to retaintheir ability to serve as a template for exponential replication by anappropriate RNA-directed RNA polymerase, as is demonstrated in theresults obtained by Lizardi et al., supra.

Replication of RNA, as opposed to target amplification using PCR, can bedone in a single step. In that step one can synthesize as many as abillion copies of the replicatable RNA that was joined to the probe inas little as twenty minutes, which theoretically could lead to detectionof a single target molecule. However, in practice the sensitivity ofthis type of probe replication is limited by the persistence ofnonspecifically bound probes. Nonspecifically bound probes will lead toreplication just as will probes hybridized to targets.

A major problem in the implementation of bioassays that employhybridization technology coupled to signal amplification systems is thebackground signal produced by nonspecifically bound probe molecules.These background signals introduce an artificial limit on thesensitivity of bioassays. In conventional bioassays this problem issometimes alleviated by the utilization of elaborate washing schemesthat are designed to remove nonspecifically bound probes. These washingschemes inevitably add to the complexity and cost of the assay.

As a means to reduce the background noise level of assays employingprobes linked to replicatable RNA by covalently joined linking moieties,European Patent Application No. 266,399 discloses what it refers to as"smart probes," that is, probes whose linked RNA is said not to serve asa template for replication unless and until the probe has hybridizedwith a target sequence. In that application two embodiments aredisclosed for smart probes.

In a first embodiment in that application, the smart probe comprises aprobe portion consisting of about 75-150 deoxynucleotides, made by invitro or in vivo methods known in the art. The smart probe alsocomprises a recombinant, replicatable RNA containing an insertedheterologous sequence of about 10-30 nucleotides, made by, e.g., themethod of Miele, E. A., Mills, D. R., and Kramer, F. R., AutocatalyticReplication of a Recombinant RNA. J. Mol. Biol. 171:281-295 (1983).Joining those two portions at their 5' ends is a linking moiety of theformula --O(PO₂)NH(CH₂)_(a) SS(CH₂)_(b) NH(PO₂)O--, where a and b areeach 2 to 20. Furthermore, the sequence at the 3' end of the DNA portionof the smart probe is capable of being (and very likely to be)hybridized to the heterologous sequence of the RNA portion of the smartprobe. The enzyme ribonuclease H is said to be capable of cleaving theRNA portion of smart probes which have not hybridized to targets, butnot be capable of cleaving the RNA portion of smart probes which havehybridized to targets, because when the probe sequence in the DNAportion of a smart probe is bound to its target, it is said to beincapable of also being hybridized to the heterologous sequence in theRNA portion of the smart probe, thereby providing a way to eliminatenonspecifically bound probes prior to amplification. Amplification viaRNA replication is said to optionally include the preliminary step ofcleaving the disulfide bond in the linking moiety.

In that embodiment, cleavage of probes not hybridized to targets is saidto be possible for ribonuclease H, because the 3' end of the DNA portionof the smart probe (which contains the probe sequence) is hybridized tothe recombinant replicatable RNA portion, presumably thereby providing asite wherein ribonuclease H can cleave the RNA and render it inoperativeas a template for amplification by an RNA-directed RNA polymerase.

In the other embodiment of a smart probe disclosed in published EuropeanPatent Application 266,399, there is a probe portion, a linking moiety,and a replicatable RNA portion, linked as described above. Here,however, the probe portion comprises not only a probe segment of 50-150nucleotides, but also additional segments, called "clamp" segments, oneither side of it, that is, a 5'-clamp segment and a 3'-clamp segment,each of about 30-60 nucleotides. Each clamp segment is said to hybridizewith a segment of the replicatable RNA portion, rendering the RNAinactive as a template for replication, unless and until the probe ishybridized with a target. That hybridization causes the clamps torelease, thereby rendering the RNA replicatable, either directly orafter optional cleavage of the disulfide bond.

The smart probes disclosed in published European Patent Application No.266,399 comprise a somewhat complicated linking moiety containing aweakly covalent and rather easily dissociable disulfide linkage.Disulfide bonds readily dissociate under reducing conditions. The twoversions of smart probes disclosed in that application rely on distantintramolecular interactions to render the probe smart. This is adisadvantage which makes such probes difficult to design, particularlysince distant interactions are not well understood. The second version,reported above, has a further complication that it utilizes two distantclamps which must displace a set of relatively strong neighboringcompliments. And, the design depends on both distant clamps hybridizingor none, which makes design very difficult.

An object of the present invention is a simple molecular allostericswitch that renders a nucleic acid hybridization probe smart, that is,capable, in an appropriate assay, of generating a signal only if theprobe is hybridized to a target sequence.

It is a further object of this invention to couple the activity of asignal generating system to the state of such a switch.

It is yet another object of this invention to develop probes containingsuch an allosteric switch that are linked to any of a number ofdifferent signal generating systems whose activity is dependent on thestate of the switch.

It is another object of this invention to develop assays of improvedsensitivity that utilize the above constructs, as well as kits forperforming such assays.

SUMMARY OF THE INVENTION

The present invention is predicated on a simple molecular allostericswitch that works on the principle that when a nucleic acid double helixis formed between a relatively short probe sequence and a targetsequence, the ends of the double helix are necessarily located at adistance from each other due to the rigidity of the double helix. Thatrigidity is discussed in detail in Shore, D., Langowski, J. and Baldwin,R. L., DNA Flexibility Studied by Covalent Closure of Short Fragmentsinto Circles, Proc. Natl. Sci. USA 78:4833-4837 (1981); and Ulanovsky,L., Bodner, M., Trifonov, E. N., and Choder, M., Curved DNA: Design,Synthesis, and Circularization, Proc. Natl. Acad. Sci. USA 83:862-866(1986).

This invention involves the use of a nucleic acid hybridization probecomprising at least the following essentials: a probe sequence ofapproximately 15-115 nucleotides in length surrounded on both sides bycomplementary nucleic acid sequences which are considerably shorter thanthe probe sequence, preferably not greatly in excess of one-half thelength of the probe sequence. This combination of three sequences formsa simple molecular allosteric switch. When not hybridized to a targetsequence, the switch secluences are hybridized to each other, which werefer to as a closed switch. When the probe sequence hybridizes to apredetermined complementary target sequence for which the probe isdesigned, the strong interaction between the probe and target sequencesto form a rigid double helix necessarily results in the dissociation ofthe switch sequences, which we refer to as an open switch. In the openconfiguration, the switch sequences are unable to interact with eachother.

The invention comprises probe molecules containing the above switchwherein one of the switch sequences, or both switch sequences incombination, comprise a biologically functional nucleic acid moietyuseful for selectively generating a detectable signal indicative of thehybridization of the probe with its predetermined target sequence.

The invention further comprises bioassay methods which take advantage ofthe allosteric change in the switch sequences in the above probemolecules to generate a detectable signal indicative of thehybridization of the probe with its predetermined target sequence. Theassay may be qualitative (a qualitative demonstration) or quantitative(a quantitative determination). It may include amplification, which maybe linear or exponential in nature.

The invention also includes kits of reagents and macromolecules forcarrying out the above bioassays.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a closed switch according to theinvention.

FIG. 2 is a schematic representation of the switch of FIG. 1, but in anopen state.

FIG. 3 is a schematic representation of the probe of Example I,containing a switch in an open state.

FIG. 4 is a schematic representation of the probe of Example II,containing a switch in an closed state.

FIG. 5 is a schematic representation of the probe of Example II,containing a switch in an open state.

FIG. 6 is a schematic representation of the probe of Example III,containing a switch in an closed state.

FIG. 7 is a schematic representation of the probe of Example III,containing a switch in an open state.

FIG. 8 is a schematic representation of the probe of Example IV,containing a switch in an closed state.

FIG. 9 is a schematic representation of the probe of Example IV,containing a switch in an open state and additionally showing aribozyme.

FIG. 10 is a detailed schematic showing the nucleotide sequences of theribozyme shown in FIG. 9.

FIG. 11 is a schematic representation of the probe of Example IV,containing a switch in an open state and additionally showing anadditional strand.

FIG. 12 is a schematic representation of the probe of Example V,containing a switch in an closed state.

FIG. 13 is a schematic representation of the probe of Example V,containing a switch in an open state.

DETAILED DESCRIPTION OF THE INVENTION

Shown in FIG. 1 is a probe, or probe portion, comprising the threeessential ingredients of a probe according to this invention, namely, aprobe sequence and complementary switch sequences on both sides of theprobe. As depicted in FIG. 1, the switch is closed. FIG. 2 is the sameprobe or probe portion in its open state.

Referring to FIG. 1, probe sequence 1 is a nucleic acid probe sequenceextending from its 5' side 2 to its 3' side 3. Immediately adjacent tothe 5' side of the probe sequence is a nucleic acid first switchsequence 4. Immediately adjacent to the 3' side of the probe sequence isa nucleic acid second switch sequence 5. Switch sequences 4 and 5 arecomplementary and hybridize to each other via hydrogen bonds 7, formingthe stem 6 of a "hairpin" secondary structure. Referring to FIG. 2,probe sequence 1 is hybridized via hydrogen bonds 9 to its predeterminedtarget sequence 8. Switch sequences 4 and 5 are apart and notinteracting with one another.

The probe may be RNA or DNA. The probe sequence 1 must be of sufficientlength to ensure a very specific interaction with its predeterminedtarget sequence 8. It should be at least about 15 nucleotides in length,although we prefer that it be at least about 20 nucleotides in length.

The probe sequence 1 should be short enough to ensure that the sides 2,3 of probe sequence 1, when hybridized to the target sequence 8 (FIG. 2)are physically prevented by the rigidity of the hybridized regionbetween sides 2 and 3 from approaching each other within a distance thatwould permit switch sequences 4, 5 from interacting with each other. Inother words, when the probe sequence is hybridized, the switch sequencesnecessarily are not hybridized to each other. An additional force helpsto drive the transition to an open state, namely, torsional forcestending to unwind stem 6 when the hybridized region shown in FIG. 2forms a double helix. In practice, the probe sequence is no longer thanabout 100 nucleotides. We prefer that the probe sequence be 20-60nucleotides in length, and most preferably, about 30 nucleotides inlength.

The switch sequences are related to the length of the probe sequence.Most preferably, we prefer that the length of the switch sequences be nomore than half the length of the probe sequence. The switch sequencesshould be at least about 10 nucleotides in length to permit formation ofa stable stem 6. Turner, D. H., Sugimoto, N., Jaeger, J. A., Longfellow,C. E., Freier, S. M. and Kierzek, R., Improved Parameters for Predictionof RNA Structure, Cold Spring Harbor Symp. Quant. Biol. 52:123-133(1987). The length of switch sequences for certain embodiments describedbelow must also be sufficiently long to contain necessary functionalsequences. We prefer switch sequences of about 10-40 nucleotides orpreferably about 10-30 nucleotides.

In designing a probe according to the invention, attention should bepaid to the relative strengths of the open switch hybrid (FIG. 2) ascompared to the closed switch hybrid (FIG. 1) under the assay conditionsto be used: the former should be greater. There are assay conditions,however, in which the strengths of hybrids is only length-dependent.Wood, W. I., Gitschier, J., Lasky, L. A., and Lawn, R. M., BaseComposition-independent Hybridization in Tetramethylammonium Chloride: AMethod for oligonucleotide Screening of Highly Complex Gene Libraries,Proc. Natl. Acad. Sci. USA 82:1585-1588 (1985).

Switch design can be readily tested by digesting probes or probeportions (FIGS. 1, 2) with appropriate nucleases before and afterhybridization to model nucleic acids containing target sequences andthen analyzing the digestion products by polyacrylamide gelelectrophoresis. This will be apparent to those skilled in the art andwill not be described further.

To help drive the transition from closed to open, one may take advantageof the principle of strand displacement to provide an additional force.Green, C., and Tibbetts, C., Reassociation Rate Limited Displacement ofDNA Strands by Branch Migration, Nucleic Acids Res. 9:1905-1918 (1981).This may be accomplished by overlapping a switch sequence with a probesequence, which means that at least one nucleotide of the switchsequence is also a nucleotide of the probe sequence.

While the switch sequences must be adjacent to the probe sequence, theyneed not be immediately adjacent to it. A few nucleotides may separatethe switch sequences from the probe sequences, but not so many that thefunctioning of the switch is materially affected, as those skilled inthe art will readily appreciate.

Probe molecules of this invention, containing the switch describedabove, can be of diverse design and still take advantage of theallosteric change that accompanies probe sequence hybridization (FIG. 2)in signal generation.

For example, a switch sequence may, by virtue of the conformation itassumes in the open state, enable an interaction with anothermacromolecule, or even a different portion of the same molecule, whichis required for the generation of a detectable signal. In Example Ibelow, the second switch sequence, in the open state, is able tohybridize with a complementary nucleic acid strand. In Example III, thefirst switch sequence, in the open state, forms a hairpin structure thatenables it to bind specifically to a viral protein. In Example IV, thesecond switch sequence, in the open state, is able to interact with anoligoribonucleotide or with an oligodeoxyribonucleotide. In Example V,the first switch sequence, in the open state, assumes a structuredconformation that enables it to interact with a relatively distantregion of the same probe molecule.

It is also possible to do the reverse. In Example II, the switchsequences can bind to a specific enzyme only when they are in the closedstate.

Signal generation using probe molecules and methods of this inventionmay vary widely. The state of the simple allosteric switch governssignal generation, which means that there is no signal generated unlessthe probe sequence hybridizes with its target sequence. We prefer signalgenerating systems that involve amplification, particularly exponentialamplification, to increase sensitivity.

The Examples which follow illustrate a few of the myriad variationsinvolving amplification. They all utilize the exponential replication ofa replicatable RNA by an RNA-directed RNA polymerase to generate areadily detectable signal. The Examples utilize MDV-1 RNA, which isdescribed in Kacian, D. L., Mills, P. R., Kramer, F. R., and Spiegelman,S., A Replicating RNA Molecule Suitable for a Detailed Analysis ofExtracellular Evolution and Replication, Proc. Nat. Acad. Sci. USA69:3038-3042 (1972). The Examples also use Q-beta replicase, which isthe specific polymerase for replicating MDV-1 RNA. Q-beta replicase isdescribed in Haruna, I. and Spiegelman, S., Specific TemplateRequirements of RNA Replicases, Proc. Nat. Acad. Sci. USA 54:579-587(1965). Any replicatable RNA and its homologous replicase could, ofcourse, be used. Other useful signal generating systems could employenzymes, enzyme cofactors, ribozymes, DNA and RNA sequences required forbiological activity (e.g., promoters, primers, or linkers required forthe ligation of plasmids used to transform bacteria). Detectable signalsare diverse and include, for example, radiation, light absorption,fluorescence, mass increase, and the presence of biologically activecompounds.

Assay techniques which can be used to detect hybridized probes of thisinvention are also diverse. In the following Examples, synthesis of areplicatable RNA is used to signal that hybridization of the probesequence has occurred. The signal generating systems illustrated in theExamples fall into three broad classes: in Examples II-III, the switchis incorporated within a replicatable RNA; in Examples IV-V, areplicatable RNA sequence is joined with a probe portion but can only bereplicated after cleavage, which is dependent upon the presence of anopen switch; and in Example I, the transcription of a replicatable RNAfrom a template added after hybridization, can only occur when an openswitch sequence forms a part of a functional promoter of transcription.

Each of the specific embodiments set forth in the accompanying Examplessatisfies the objective of generating a signal only if the probe ishybridized to a target sequence. Either the biological activity of thesignal generating systems illustrated depends strictly on the state ofthe switch, or the state of the switch provides a means for renderingnonspecifically bound probes unable to generate signals, or the state ofthe switch provides a means for separating hybridized probes fromnonspecifically bound probes. Thus, each of the specific embodimentsmarkedly reduces the background caused by nonspecifically bound probes,thereby significantly improving the sensitivity of the assays, includingassays which include amplification.

EXAMPLE I

In this example, the probe is a single DNA strand designed to containthree sequences: a probe sequence approximately 34 nucleotides inlength; a first switch sequence of about 17 nucleotides immediatelyadjacent to the 5' side of the probe sequence; and a second switchsequence of about 17 nucleotides immediately adjacent to the 3' side ofthe probe sequence. The switch sequences are designed to becomplementary to one another. When hybridized to each other, thehybridized switch sequences comprise a promoter for the DNA-directed RNApolymerase, bacteriophage T7 RNA polymerase. In this application, werefer to the first switch sequence as a "promoter sequence" and thesecond switch sequence as a "promoter-complement" sequence. In thisexample, the switch sequences comprise the ends of the probe molecule.The design of promoter and promoter-complement sequences is according toOsterman, H. L. and Coleman, J. E., "T7 Ribonucleic AcidPolymerase-Promoter Interactions," Biochemistry 20:4885-4892 (1981). Theparticular promoter-complement sequence we have chosen to work with isTAATACGACTCACTATA.

The probe molecule, including a probe sequence complementary to apredetermined target sequence, can be made by chemical synthesis ofoligodeoxyribonucleotides using methods well known in the art, e.g.,Gait, M. J., OLIGONUCLEOTIDE SYNTHESIS, IRL Press, Oxford, UnitedKingdom (1984).

The probe of this example can be used to detect a DNA or RNA targetsequence which is complementary to the probe sequence. The targetsequence may be in a sample containing other, unrelated nucleic acidsand other materials, for example, proteins. The probe may be used todetect a gene segment of an infectious agent (virus, bacterium,protozoan, etc.) in a clinical sample of, for example, human blood orurine.

The target sequence must be exposed to the probe. This is done bytechniques well known to the art. Commonly, but not necessarily, nucleicacid is isolated from a sample before the probe is added.

The probe and the sample, which may contain nucleic acid targetsequences, are next incubated under conditions, including time andtemperature, appropriate to cause hybridization of probe sequences withtarget sequences. Appropriate conditions are well known in the art. Forquantitative determination of the number of target sequences present, anamount of probe in excess, preferably in substantial excess, of thehighest anticipated target amount should be used. If only a qualitativedemonstration of the presence of target sequences is desired, a lesseramount of probe can be used.

Probes hybridized to targets are separated from unbound probes bymethods well known to the art, for example, through the use of captureprobes.

After separation, the treated sample will contain probes hybridized totargets (FIG. 2) and also nonspecifically bound probes. The two are notin the same form, however. In the hybridized probes the allostericswitches are open; that is, the switch sequences are not hybridized toeach other. In the nonspecifically bound probes, however, the switchsequences remain hybridized to each other.

Detecting those probes with open switches will now be described. Thisexample includes amplification prior to detection.

Referring to FIG. 3, the sample is incubated with a single-stranded DNAmolecule 10 comprising a promoter sequence 11 and a template sequence 12for the transcription of a replicatable RNA. The promoter sequence 11allows hybridization via hydrogen bonds 13, under conditions known tothe art, to the promoter-complement of the second switch sequence 5 ofprobes having open switches. Specifically, this DNA molecule consists ofthe 17 deoxyribonucleotides of the promoter sequence (complementary tothe promoter-complement set forth above) followed by the 244deoxyribonucleotides complementary to MDV-poly (+) RNA described inLizardi et al., supra. This DNA molecule can be prepared by isolatingone of the complementary strands of a suitable restriction fragment of aplasmid containing that sequence by methods known in the art. Maniatis,T., Fritsch, E. F., and Sanbrook, J., MOLECULAR CLONING: A LABORATORYMANUAL Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982).The suitable plasmid that we constructed contained (1) a uniquerestriction site (that is, one contained nowhere else in the plasmid)upstream from and close to the promoter, and (2) and Sma I restrictionsite at the end of the MDV-Poly CDNA sequence distal to the promoter.

Subsequently, the sample is incubated with commercially available clonedbacteriophage T7 RNA polymerase in order to synthesize about 50-200, ormore, MDV-poly RNA transcripts for each open switch, using conditionsknown to the art. Milligan, J. F., Duncan, R. G., Witherell, G. W. andUhlenbeck, O. C., Oligoribonucleotide Synthesis Using T7 RNA Polymeraseand Synthetic DNA Templates, Nucleic Acids Research 15:8783-8798 (1987).

Then, Q-beta replicase, an RNA-directed RNA polymerase, is added andincubated with the MDV-poly RNA transcripts, which are templates forthat polymerase. We prepared Q-beta replicase by the method of Eoyang,L., and August, J. T., Q-beta RNA polymerase from phage Q-beta-infectedE. coli, pp. 829-839. In: Procedures in Nucleic Acid Research, Volume 2.(Cantoni, G. L., and Davis, D. R., eds.). Harper and Row, New York(1971). Incubation is carried out under conditions suitable forexponential amplification of the transcripts. Kramer, F. R., Mills, D.R., Cole, P. E., Nishihara, T., and Spiegelman, S., Evolution in vitro:Sequence and Phenotype of a Mutant RNA Resistant to Ethidium Bromide. J.Mol. Biol. 89:719-736 (1974).

Detection of the exponentially amplified RNA can be done by any of avariety of physical and chemical means, as described earlier in thisapplication. For a quantitative determination, the amount of RNAdetected after a fixed time of incubation with the RNA-directed RNApolymerase is a measure of the number of target sequence present in thesample.

EXAMPLE II

Referring to FIG. 4, in this example, the probe is a replicatablerecombinant RNA 14. Miele, E. A., Mills, D. R., and Kramer, F. R.,Autocatalytic Replication of a Recombinant RNA, J. Mol. Biol.171:281-295 (1983). It may be prepared according to the method ofLizardi et al., supra. For purposes of preparing a probe according tothis example, the heterologous sequence 15 contained within thereplicatable recombinant RNA is designed to contain three sequences: aprobe sequence 16 approximately 46 nucleotides in length; a first switchsequence 17 of about 23 nucleotides immediately adjacent to the 5' sideof the probe sequence; and a second switch sequence 18 of about 23nucleotides immediately adjacent to the 3' side of the probe sequence.The switch sequences are designed to form a double-stranded recognitionsite for Escherichia coli ribonuclease III when hybridized to eachother. This recognition site will not be present when the switchsequences are not hybridized to each other. The particular recognitionsite we use is shown in FIG. 4 and is described by Rosenberg, M. andKramer, R. A., Nucleotide Sequence Surrounding a Ribonuclease IIIProcessing Site in Bacteriophage T7 RNA, Proc. Natl. Acad. Sci. USA74:984-988 (1977). It can be made by transcription from a recombinantplasmid utilizing techniques described in Lizardi et al., supra.

Exposure of the target sequence, hybridization of the probe with thetarget sequence, and separation from unbound probes, are as described inExample I. As shown in FIG. 5, probe sequence 16 of a hybridized probe14 is hybridized to target sequence 8, thereby forcing apart switchsequences 17, 18.

The sample is then incubated with E. coli ribonuclease III underappropriate conditions known to the art to cleave all thenonspecifically bound probes (and any unbound probes which may remain),rendering them unable to serve as templates for exponential replicationby Q-beta replicase. Nishihara, T., Mills, D. R., and Kramer, F. R.,Localization of the Q-beta Replicape Recognition Site in MDV-1 RNA, J.Biochem. 93:669-674 (1983). The ribonuclease III is then removed fromthe sample by methods, e.g., phenol extraction, well known in the art.

We release the probe from the target sequence by a brief heating step,Lizardi et al. supra, although preliminary experiments have indicatedthat this step may be optional.

Exponential replication of the probe by Q-beta replicase and detectionproceed as described in Example I.

EXAMPLE III

In this example the probe 19 (FIG. 6) is a replicatable recombinant RNAas in Example II, except that the probe sequence 20 is about 38nucleotides in length and that the complementary switch sequences 21,22, of about 19 nucleotides, are designed such that when they arehybridized to one another they do not form a binding site for the coatprotein of bacteriophage R17, but when not so hybridized, as shown inFIG. 7, the first switch sequence 21 organizes so as to comprise asecondary structure which is a strong binding site for that coatprotein. Carey, J., Cameron, V., de Haseth, P. L. and Uhlenbeck, O. C.,Sequence-Specific Interaction of R17 Coat Protein With Its RibonucleicAcid Binding Site, Biochemistry 22:2601-2610 (1983).

Exposure of the target sequence, hybridization of the probe with thetarget sequence, and separation from unbound probes are as described inExample I.

The bacteriophage R17 coat protein is covalently linked to a solidsupport, such as, for example, Sephadex or Sepharoic beads, magneticbeads, or microtiter plates, by methods well known in the art. Anexample of such a method of linkage is described in Alagon, A. J., andKing, T. P., Activation of Polysaccharides with 2-Iminothiolane and ItsUses, Biochemistry 19:4331-4345 (1980). The washed sample, containingprobes bound to target sequences and nonspecifically bound probes, isadded to the insolubilized R17 coat protein. Nonspecifically boundprobes are removed by washing.

We release the probe from both the R17 coat protein and the targetsequence by a brief heating step, and remove the solid support.

Exponential replication of the probe by Q-beta replicase and detectionproceed as described in Example I.

EXAMPLE IV

In this example, the probe 23 (FIG. 8) is a single strand of RNAdesigned to contain four functionally distinct sequences: a probesequence 24 approximately 34 nucleotides in length; a first switchsequence 25 of about 17 nucleotides immediately adjacent to the 5' sideof the probe sequence; a second switch sequence 26 complementary to, andof the same length as, the first and located immediately adjacent to the3' side of the probe sequence; and a replicatable RNA sequence 27extending from the 3' side of the second switch sequence, wherein atleast five nucleotides of said replicatable RNA sequence are alsonucleotides of the 3' side of the second switch sequence; that is, thereplicatable RNA sequence can be considered to overlap the second switchsequence.

Exposure of the target sequence, hybridization of probes to targetsequences and separation of unbound probes are performed underappropriate conditions known to the art, as in Example I. As shown inFIG. 9, probe sequence 24 is hybridized to target sequence 8, and switchsequences 25, 26 are forced apart, thereby freeing replicatable RNAsequence 27. The replicatable RNA sequences of bound probes are, at thispoint, not subject to exponential replication by RNA polymerase eventhough the switches are open. The replicatable RNA sequences 27 must becleaved at their 5' sides to render them subject to exponentialreplication. Nishihara, T., Mills, D. R., and Kramer, F. R.,Localization of the Q-beta Replicase Recognition Site in MDV-1 RNA, J.Biochem. 93:669-674 (1983).

There are two means, at least, to cleave the replicatable RNA sequences.One is ribozyme cleavage. Another is cleavage by ribonuclease H. Weprefer the former, which will be described first.

A. Ribozyme Cleavage

Ribozymes are structured RNA molecules that are capable of catalyzing achemical reaction, such as particularly the cleavage of a phosphodiesterbond. It is well known in the art that a ribozyme can be constructed bythe interaction of two separate oligribonucleotides, one of which iscleaved at a particular phosphodiester bond when incubated under known,appropriate conditions. Uhlenbeck, O. C., A Small CatalyticOligoribonucleotide, Nature 328:590-600 (1987); Haseloff, J. andGerlach, W. L., Simple RNA Enzymes with New and Highly SpecificEndoribonuclease Activities, Nature 334:585-591 (1988).

The requirements for the two segments of an active ribozyme are outlinedin the two references cited above. For purposes of this invention, thesecond switch sequence of our probe is designed to satisfy therequirements of the sequence that is cleaved. The replicatable RNAsequence with which we have chosen to proceed is MDV-poly (+) RNAaccording to Lizardi et al. supra. Our preferred design is shown in FIG.8. As shown there, the second switch sequence is 17 nucleotides inlength, and 11 nucleotides of the 5' side of the MDV-poly (+) RNA arealso nucleotides of the 3' side of the second switch sequence. Thesecond switch sequence includes the required GUC sequence needed forcleavage of the phosphodister bond on the 3' side of the GUC sequence,that is, on the 5' side of the replicatable RNA sequence. In designingthe second switch sequence, care is taken to ensure that the subsequenthybridization to form the ribozyme will be more likely to occur than theinteraction that can occur between the sides of the replicatable RNAsequence.

The probe can be made by transcription from a suitable recombinantplasmid. Such a plasmid is designed utilizing methods known to the artwith the criteria of Lizardi et al., supra. It is constructed by methodswell known to the art. Maniatis, T., Fritsch, E. F., and Sambrook, J.,MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1982).

The non-cleaved strand 28, which is capable of forming the requiredribozyme, is also shown in FIG. 9. It is made by methods well known tothe art. Milligan, J. F., Duncan, R. G., Witherell, G. W. and Uhlenbeck,O. C., Oligoribonucleotide Synthesis Using T7 RNA Polymerase andSynthetic DNA Templates, Nucleic Acids Research 15:8783-8798 (1987).FIG. 10 shows the nucleotide sequences of the ribozyme formed by switchsequence 26 and strand 28 of FIG. 9.

Following separation of unbound probes, which we prefer, the non-cleavedstrand 28 described above is incubated with the sample under conditions,known to the art, that will promote hybridization of that strand withthe second switch sequence in probes hybridized to target sequences toform the desired ribozyme. Incubation under known conditions referred toabove cleaves the replicatable RNA from those probes and permitsreplicatable RNA to serve as a template for exponential replication byQ-beta replicase. Referring to FIG. 10, cleavage occurs in strand 26between the sixth and seventh nucleotides from the left as shown in thefigure. Exponential replication and detection proceed as described inExample I.

B. Ribonuclease H Cleavage

The probe for this embodiment may be identical to the probe shown inFIG. 8 and described above. In this embodiment we use commerciallyavailable E. coli ribonuclease H, which cleaves an RNA strand when it ishybridized to a short DNA oligonucleotide within the hybridized region.Donis-Keller, H., Site Specific Enzymatic Cleavage of RNA, Nucleic AcidsRes. 7:179-192 (1979).

To take advantage of this, we synthesize a short DNA oligonucleotide 29(FIG. 11) of about 12 nucleotides that will hybridize to the secondswitch sequence on both sides of the GUC sequence.

Following separation of unbound probes, which we prefer, the short DNAoligonucleotide 29 is incubated with the sample under well knownconditions that will promote its hybridization (FIG. 11) to the secondswitch sequence. Then the ribonuclease H is added to catalyze cleavageduring an incubation under known conditions. (Donis-Keller, supra).Exponential replication by Q-beta replicase and detection proceed asdescribed in Example I.

EXAMPLE V

This example resembles Example IV-A except that the ribozyme sequencesare both part of the probe. The probe 30 (FIG. 12) is a single-strandedRNA, prepared as described in Example IV but designed to contain fivesequences: a probe sequence 31 approximately 34 nucleotides in length; afirst switch sequence 32 of about 17 nucleotides having the sequence ofthe non-cleaved strand 28 shown in FIG. 9; a second switch sequence 33of about 17 nucleotides complementary to the first sequence, as inExample I; a spacer sequence 34 of approximately 45 nucleotidesextending from the 3' side of the second switch sequence, and areplicatable RNA moiety 35. The six nucleotides at the 3' side of thespacer sequence are identical to the six nucleotides at the 5' side ofthe second switch sequence shown in FIG. 9. Thus, the region in whichthe spacer sequence is joined to the replicatable RNA sequence comprisesthe cleavable strand of a ribozyme, just as does the second switchsequence 26 in Example IV-A. In the unbound probe, the first switchsequence 32 is hybridized to the second switch sequence 33. In probeshybridized to target sequences, where the switch is open, however, thefirst switch sequence 32 is available to hybridize with the region inwhich the 3' side of the spacer sequence 34 is joined to the 5' side ofthe replicatable RNA sequence 35, thereby forming a ribozyme. The spacer34 is designed to be long enough to permit that hybridization.

Exposure of the target sequence, hybridizing of probes to targetsequences, and separation of unbound probes, which we prefer, is asdescribed in Example I. Upon hybridization of a probe to a targetsequence (FIG. 13), the switch sequences 32, 33 are not hybridized toeach other and the ribozyme 36 is formed.

Release of the replicatable RNA, exponential replication and detectionproceed as in Example IV-A.

As stated above the assays of this invention may be qualitative orquantitative. As one skilled in the art will readily appreciate, for aqualitative demonstration of a predetermined target sequence by themethods described above, biological and chemical reagents used in theassays must be used in readily determinable quantities sufficient togenerate a reproducible, detectable signal in a sensitive assay.

For a quantitative determination, the amount of probe added should besubstantially in excess of the highest amount of target sequenceexpected and incubation should be carried out under conditions such thatvirtually all target sequences hybridize with probes. By "virtually all"we mean a very high percentage sufficient to impart reproducibility tothe assay. In subsequent steps through signal detection, each stepshould be similarly quantitative. For example, destruction of unboundprobes should destroy virtually all of the unbound probes forreproducibility and also to eliminate background noise. Transcriptionand replication steps should utilize sufficient reagents to bequantitative and should be carried out for set times for the sake ofreproducibility.

Often, both qualitative and quantitative assays will include parallelassays of at least a negative control, that is, one not containingtarget sequence, and at times will also include a series of samplescontaining known amounts of target sequence, such as a geometricallyincreasing series.

The present invention is also directed to assay kits useful for thequalitative detection or quantitative determination of at least onespecific, predetermined nucleic acid target sequence using probemolecules of this invention. Assay kits will include quantities of oneor more probes which comprise at least the three essential sequencesdescribed above and at least one additional biologically activemolecule, for example, a DNA strand, a ribozyme former, an RNA strand oran enzyme, useful for generating a signal indicative of switch opening.Kits may also include additional reagents such wash solutions,insolubilizing reagents and materials, amplification reagents anddetection reagents. Amplification reagents may include enzymes andnucleotides. Detection reagents may include labeled nucleotides andcolor-forming substrates. Kits designed for research may includeplasmids which will enable a researcher to prepare probes according tothis invention containing any desired probe sequence.

We claim:
 1. A method for the detection of at least one predeterminednucleic acid target sequence in a sample containing nucleic acid andcomprising the steps of:a) adding to the sample hybridization probescomprising a molecular switch consisting essentially of threeoligonucleotide sequences:i) a probe sequence of from about 20 to about60 nucleotides, having a 5' side and a 3' side, which probe sequence iscomplementary to said target sequence, ii) a 5' sequence of from about10 to about 40 nucleotides, including a first switch sequence of atleast about 10 nucleotides, immediately adjacent to and linked to the 5'side of the probe sequence, and iii) a 3' sequence of from about 10 toabout 40 nucleotides, including a second switch sequence of at leastabout 10 nucleotides, immediately adjacent to and linked to the 3' sideof the probe sequence, said second switch sequence being hybridized withsaid first switch sequence only when said probe sequence is nothybridized to said target sequence, wherein said molecular switchincludes a preselected nucleic acid sequence for selectively generatinga detectable signal when the probe sequence is hybridized with saidtarget sequence, b) specifically hybridizing probes with said targetsequence, c) destroying the ability of probes which did not hybridizespecifically with said target sequence in step b to generate a signal,d) generating a signal from probes which did hybridize specifically withsaid target sequence in step b, and e) detecting the signal in order todetect the predetermined nucleic acid target sequence.
 2. A methodaccording to claim 1 wherein said probe is a replicatable RNA.
 3. Amethod according to claim 2 wherein step c comprises destroying with aribonuclease the replicability of probes which did not hybridizespecifically with said target sequence and wherein step d comprisesexponentially replicating probes which did hybridize specifically withsaid target sequence.
 4. A method according to claim 2 wherein probeshybridized specifically with said target sequence in step b areseparated from probes which did not so hybridize prior to performingstep d.
 5. A method according to claim 1 comprising a quantitativedetermination of the amount of said target sequence in said sample,wherein the amount of probe added in step a substantially exceeds themaximum number of target sequences expected in said samples, whereinstep b proceeds until virtually all target sequences hybridize withprobes, wherein step c the replicability of virtually all of the probeswhich did not hybridize specifically with said target sequence isdestroyed, wherein step d is carried out for a predetermined time, andwherein step e is quantitative.
 6. A method according to claim 5additionally comprising performing a parallel assay on a sample whichdoes not contain said target sequence.
 7. A method for the detection ofat least one predetermined nucleic acid target sequence in a samplecontaining nucleic acid comprising the steps ofa) adding to the samplehybridization probes comprising a molecular switch consistingessentially of three oligonucleotide sequence:i) a probe sequence offrom about 20 to about 60 nucleotides, having a 5' side and a 3' side,which probe sequence is complementary to said target sequence, ii) a 5'sequence of from about 10 to about 40 nucleotides, including a firstswitch sequence of at least about 10 nucleotides, immediately adjacentto and linked to the 5' side of the probe sequence, and iii) a 3'sequence of from about 10 to about 40 nucleotides, including a secondswitch sequence of at least about 10 nucleotides, immediately adjacentto and linked to the 3' side of the probe sequence, said second switchsequence being hybridized with said first switch sequence only when saidprobe sequence is not hybridized to said target sequence, wherein saidmolecular switch includes a preselected nucleic acid sequence forselectively generating a detectable signal when the probe sequence ishybridized with said target sequence, said detectable signal resultingfrom exponential replication of replicable RNA, b) specificallyhybridizing probes with said target sequence, c) exponentiallyreplicating the replicatable RNA, and d) detecting the replicationproducts in order to detect the predetermined nucleic acid targetsequence.
 8. A method according to claim 7, wherein said probe is afirst DNA strand, comprising the additional step of hybridizing to thesecond switch sequence of said probe a second DNA strand which is atemplate for the transcription of a replicatable RNA prior to step c. 9.A method according to claim 7, wherein said probe is a replicatablerecombinant RNA, comprising the additional step of separating probeswhich hybridized with target sequence in step b from probes which didnot so hybridize prior to step c, and wherein the replicatable RNA ofstep c is said replicatable recombinant RNA.
 10. A method according toclaim 7 wherein said probe is an RNA strand comprising a replicatableRNA sequence extending from said second switch sequence, comprising theadditional step of selectively cleaving said replicatable RNA sequencefrom probes which hybridized with target sequences in step b prior tostep c, and wherein the replicatable RNA of step c is said replicatableRNA sequence.
 11. A method according to claim 10 wherein the step ofcleaving involves ribozyme cleavage.
 12. A method according to claim 10wherein the step of cleaving includes adding a ribonuclease to thesample.
 13. A method according to claim 7 comprising a quantitativedetermination of the amount of said target sequence in said sample,wherein the amount of probe added in step a substantially exceeds themaximum number of target sequences expected in said sample, wherein stepb proceeds until virtually all target hybridize with probes, whereinstep c is carried out for a predetermined time, and wherein step d isquantitative.
 14. A method according to claim 13 additionally comprisingperforming a parallel assay on a sample which does not contain saidtarget sequence.
 15. A method for the detection of at least onepredetermined nucleic acid target sequence in a sample containingnucleic acid comprising the steps ofa) adding to the samplehybridization probes comprising a molecular switch consistingessentially of three oligonucleotides sequences:i) a probe sequence offrom about 20 to about 60 nucleotides, having a 5' side and a 3' side,which probe sequence is complementary to said target sequence, ii) a 5'sequence of from about 10 to about 40 nucleotides, including a firstswitch sequence of at least about 10 nucleotides, immediately adjacentto and linked to the 5' side of the probe sequence, and iii) a 3'sequence of from about 10 to about 40 nucleotides, including a secondswitch sequence of at least about 10 nucleotides, immediately adjacentto and linked to the 3' side of the probe sequence, said second switchsequence being hybridized with said first switch sequence being whensaid probe sequence is not hybridized to said target sequence, whereinsaid molecular switch includes a preselected nucleic acid sequence forselectively generating a detectable signal from an RNA signal generatorsequence of said hybridization probe when the probe sequence ishybridized with said target sequence,b) specifically hybridizing probeswith said target sequence, c) cleaving said RNA-signal generatorsequence from probes which hybridized with target sequences in step b,d) generating an amplified signal using said RNA-signal generator, ande) detecting said amplified signal in order to detect the predeterminednucleic acid target sequence.
 16. A method according to claim 15 whereinsaid RNA-signal generator includes an enzyme which generates a colorsignal.
 17. A test kit for performing an assay according to claim 1comprising a quantity of said probes and an appropriate RNA replicase.18. A test kit for performing an assay according to claim 7 comprising aquantity of said probes and an appropriate RNA replicase.
 19. The methodof claim 8 wherein the replicatable RNA is MDV-poly-RNA.
 20. The methodof claim 8 wherein the second DNA strand includes a promoter sequencefor a DNA directed RNA polymerase.
 21. The method of claim 20 whereinthe exponential replication of step (c) is performed with Q-betareplicase.
 22. The method of claim 9 wherein the predetermined targetsequence is a gene segment.
 23. The method of claim 20 wherein thepromoter is for T7 RNA polymerase.